The invention is in the field of bioorganic chemistry, more specifically the field of conjugation of biomolecules. The conjugated products prepared by the methods of the invention are useful, for example, as inoculants for the generation of antibodies, and as vaccines. The methods of the invention may also be used to immobilize biomolecules on solid supports. The immobilized biomolecules are useful in many fields, such as for example catalysis, separation, analysis, and diagnostics.
The conjugation of biomolecules to solid and gel supports is a common operation in many laboratories, and many methods have been developed for this purpose. Immobilization of enzymes (I. Chernukhin, E. Klenova, Anal. Biochem. 2000, 280:178-81), oligonucleotides (J. Andreadis; L. Chrisey, Nucleic Acids Res. 2000, 28:e5; A. Drobyshev et al., Nucl. Acids. Res. 1999, 27:4100-4105), antibodies (P. Soltys, M. Etzel, Biomaterials 2000, 21:37-48), and antigens (M. Oshima, M. Atassi, Immunol. Invest. 1989, 18:841-851) on solid and gel supports enables the preparation of useful products such as chromatographic media (Meth. Enzym., W. Jakoby, M. Wilchek, eds., 1974, 34, Academic Press, NY), catalysts (T. Krogh et al., Anal. Biochem., 1999, 274:153-62), biosensors (J. Spiker, K. Kang, Biotechnol. Bioeng. 1999, 66:158-63), and numerous diagnostic (G. Ramsay, Nature Biotechnol., 1998, 16:40-44) and research tools (C. Bieri et al., Nature Biotechnol. 1999, 17:1105-1108). Even whole cells may be immobilized by such methods (E. Olivares, W. Malaisse, Int. J. Mol. Med. 2000, 5:289-290).
The most robust form of attachment of a biomolecule to a surface or other support is via covalent bonds. Typically, such bonds are heteroatom-based (e.g., amide, ester, and disulfide bonds), because such bonds are easily formed under mild conditions. Non-covalent attachment via specific binding pairs (e.g., biotin-avidin or antibody-antigen interactions) is also commonly employed, but such methods still require initial conjugation of the specific binding pairs to the biomolecule and support. The use of carbon-carbon bonds for this purpose is very rare, because formation of Cxe2x80x94C bonds is more difficult, especially under the mild aqueous conditions appropriate for working with proteins.
The use of the Diels-Alder reaction to attach a member of a specific binding pair has been described. In this report (M. N. Yousaf and M. Mrksich, J. Am. Chem. Soc., 1999, 121:4286), a Diels-Alder reaction was used to covalently attach a biotinylated diene to an immobilized dienophile, and the immobilized biotin was subsequently used to non-covalently immobilize streptavidin. These workers have more recently used a Diels-Alder reaction to immobilize the peptide RGDS on a self-assembled alkanethiol monolayer on a gold surface (M. N. Yousaf, B. T. Houseman, M. Mrksich, Angew. Chem. Int. Ed. Engl., 2001, 40:1093). The use of the Diels-Alder reaction to effect the actual covalent coupling or immobilization event of large biomolecules, however, had not previously been described.
The conjugation of biomolecules to one another is likewise a very common procedure, and is subject to most of the concerns and limitations described above for biomolecule immobilization. Covalent attachment of haptens to proteins has been a target of synthetic endeavors since the discovery by Landsteiner that this process can convert non-immunogenic molecules to immunogenic materials (K. Landsteiner, H. Lampl, Biochem. Zeitschr. 1918, 86:343). The application of this concept to carbohydrates by Goebel and Avery revealed that covalent carbohydrate-protein conjugates are immunogenic and can generate anti-carbohydrate antibodies (W. Goebel, J. Exp. Med. 1940, 72:33). The use of Landsteiner""s principle has led to the development of carbohydrate-protein conjugates that are valuable tools in glycomedical research, and that are useful as pharmaceuticals. In particular, protein conjugates of fragments of the capsular polysaccharide of Haemophilus influenzae type b have become established as successful vaccines (J. Robbins et al., J. Am. Med. Assoc. 1996, 276:1181). Several other bacterial saccharide-protein conjugates are in various stages of clinical studies (E. Konadu et al., J. Infect. Dis. 1998, 177:383-387; E. Konadu et al., Infect. Immun. 2000, 68:1529-1534) while numerous others are in the pre-clinical phase (V. Pozsgay et al., Proc. Natl. Acad. Sci. USA 1999, 96:5194).
The choice of methods for covalent bond formation between biomolecules such as carbohydrates and proteins is restricted by their limited solubility in organic solvents, and in many cases by their pH and temperature sensitivity. In almost all cases, water is the only solvent that can be used for conjugation of carbohydrates or proteins, and the conditions are usually limited to temperatures under 50xc2x0 C. and pH values between 6 and 8.
Numerous methods have been developed for the attachment of polysaccharides to proteins (C. Peeters et al., in Vaccine Protocols, A. Robinson et al, Eds., 1996Humana Press, NJ, p. 111; W. Dick, Jr., M. Beurret, in Contrib. Microbiol. Immunol., J. Cruse and R. Lewis, eds., 1989, 10:48-114, Karger, Basel; H. Jennings, R. Sood, in Neoglycoconjugates. Preparation and Applications, Y. Lee, R. Lee, eds., Academic Press, New York, 1994, p. 325). However, only a few of these methods are capable of coupling oligosaccharides to carriers in a site-selective fashion. Most prominent among these is reductive amination, which converts the reducing-end residue of the polysaccharide into a polyhydroxy alkylamino moiety, which unfortunately causes the loss of this unit as a true carbohydrate in the resulting glycoconjugate (V. Pozsgay, Glycoconjugate J. 1993, 10:133).
This problem can be solved by chemical synthesis of oligosaccharide glycosides with aglycons that bear a (latent) reactive group. Examples include alkenyl groups (M. Nashed, Carbohydr. Res. 1983, 123:241-246; J. Allen, S. Danishefsky, J. Am. Chem. Soc. 1999, 121:10875), 3-aminopropyl (G. Veeneman et al., Tetrahedron 1989, 45:7433), 4-aminophenylethyl (R. Eby, Carbohydr. Res. 1979, 70:75), 4-aminophenyl (S. Stirm et al., Justus Liebigs Ann. Chem. 1966, 696:180), 6-aminohexyl (J. Hermans et al., Rec. Trav. Chim. Pays-Bas 1987, 106:498; R. Lee et al., Biochemistry 1989, 28:1856), 5-methoxycarbonylpentyl (S. Sabesan, J. Paulson, J. Am. Chem. Soc. 1986, 108:2068; V. Pozsgay, Org. Chem. 1998, 63:5983), 8-methoxycarbonyloctyl (R. Lemieux et al., J. Am. Chem. Soc. 1975, 97, 4076; B. Pinto et al., Carbohydr. Res. 1991, 210, 199) 4-aminobenzyl (W. Goebel, J. Exp. Med. 1940, 72:33), xcfx89-aldehydoalkyl (V. Pozsgay, Glycoconjugate J. 1993, 10:133), 3-(2-aminoethylthio)propyl (Y. Lee, R. Lee, Carbohydr. Res. 1974, 37:193), 2-chloroethylthioglycosides (M. Ticha et al., Glycoconjugate J. 1996, 13:681) and 1-O-succinimide derivatives (M. Andersson, S. Oscarson, Bioconjugate Chem. 1993, 4:246; B. Davis, J. Chem. Soc. Perkin I 1999, 3215).
These aglycons introduce spacers that can be linked to a protein either directly or after insertion of a secondary linker. For this purpose the use of an activated dicarboxylic acid has been reported (R. van den Berg et al., Eur. J. Org. Chem. 1999, 2593-2600). In another procedure, a sulfhydryl group at the terminal position of the spacer allows the formation of a disulfide bridge with proteins using the dithiopyridyl method (J. Evenberg et al., J. Infect. Dis. 1992, 165(sup. 1):S152). In a related protocol, a thiolated protein is coupled with a maleimido-derivatized saccharide (J. Mahoney, R. Schnaar, Methods Enzymol. 1994, 242:17). N-acryloylamidophenyl glycosides may be coupled to unmodified proteins using a Michael addition (A. Romanowska et al., Methods Enzymol. 1994, 242:90). As an alternative to glycoside formation, direct coupling of a carbohydrate to a linker via amide bonds has also been used (A. Fattom et al., Infect. Immun. 1992, 60:584-589), but this approach is limited to carboxyl-containing carbohydrates.
The yields of any of these methods rarely exceed 40%, and are generally in the 10-20% range (R. van den Berg et al., Eur. J. Org. Chem. 1999, 2593-2600), especially when medium or high carbohydrate loading in the conjugate is attempted. This problem is compounded by the fact that the oligosaccharide haptens, usually obtained in multistep syntheses or by controlled degradation of polysaccharides, can rarely be recovered in their active or activable form after the coupling procedure. An additional problem with most chemical coupling methods employed to date is the formation of cross-linked byproducts, due to the presence of multiple reactive functional groups (e.g., amines, acids, hydroxyls, and sulfhydryls) on most biomolecules. Avoidance of this problem requires that the reactive groups be blocked, which requires additional processing steps and may alter the physicochemical and immunological properties of the biomolecule. Thus, there remains a need for a mild and site-selective method for coupling biomolecules to one another, which avoids the problems of low yields, crosslinking, and loss of starting materials. For similar reasons there remains a need for mild and selective methods for attaching biomolecules to surfaces and solid and gel supports.
The present invention provides an experimentally simple protocol for the covalent attachment of biomolecules to one another and to supports, that can avoid many of the above-mentioned problems. The invention makes use of the well-known Diels-Alder cycloaddition reaction that takes place between a double bond and a conjugated diene. This reaction has traditionally been carried out in organic solvents, but can proceed in aqueous solutions as well (R. Breslow, D. Rideout, J. Am. Chem. Soc. 1980, 102:7816; A. Lubineau, J. Auge, Top. Curr. Chem. 1999, 206:1; P. Garner, in Organic Synthesis in Water, P. Grieco, ed., Blackie Academic and Professional, London, 1998, p. 1.)
Carbohydrates have been employed as chiral auxiliaries and/or water solubilizing agents for Diels-Alder reactions, wherein a conjugated diene system is converted to a glycoside prior to the cycloaddition (A. Lubineau et al., J. Chem. Soc. Perkin 11997, 2863-2867; see also S. Pellegrinet, R. Spanevello, Org. Lett. 2000, 2:1073-1076). As noted above, the Diels-Alder reaction has also been used to covalently attach biotin to a support (M. N. Yousaf and M. Mrksich, J. Am. Chem. Soc., 1999, 121:4286). However, the Diels-Alder reaction has not previously been extended to the direct covalent conjugation of biopolymers or other types of polymeric materials. Among the advantages of the method of the invention are the mild and neutral conditions, good yields, negligible cross-linking, and facile recovery of excess and/or unreacted biomolecules in their conjugatable form.
The invention also provides conjugated biomolecules, which are useful as immunostimulatory agents for production of antibodies and induction of immunity, methods of inducing antibody production with the conjugated biomolecules, and vaccine compositions comprising the conjugated biomolecules.
The invention also provides polyclonal and monoclonal antibodies generated by administration of the conjugated biomolecules to a mammal, and methods of using the induced antibodies for inducing passive immunity. The antibodies are useful of therapeutic, diagnostic, and analytical purposes.
The invention also provides immobilized biomolecules, and methods for their preparation, which are useful in many areas, such as chromatographic media, catalysts, components of diagnostic devices, biosensors, and as research tools.